This invention relates generally to a method for effectively clearing turbidity in a biological sample such as human serum and to a reagent used for that purpose. More particularly, it relates to a reagent comprised of a surfactant and an enzyme and to its application as a turbidity-removing agent.
Turbidity in a biological sample can cause severe problems. It results in poor or incorrect readings and therefore highly questionable determinations.
Turbidity in serum and plasma samples is usually a serious problem in clinical photometric analysis. It gives false data and often yields misleading photometric determinations of serum ingredients. The turbidity is believed caused primarily by the elevation of triglycerides in serum of patients having hyperlipoproteinemia with or without elevated total cholesterol. Abnormal elevation of cholesterol in serum has been shown to correlate with a high risk artherosclerosis. Determination of cholesterol and triglycerides is important since accurate data will help the doctor to diagnose patients with hyperlipoproteinemia and to predict certain heart disease. Other tests such as aspartate aminotransferase (GOT), alanine aminotransferase (GPT) and lactate dehydrogenase (LDH), etc. have also suffered from the same difficulties as cholesterol or triglyceride determination, as mentioned above, when turbid samples are examined.
Clinical tests of turbid serum has been handled by treating samples with high concentration of surfactants such as polyoxyethylated lauric acid (U.S. Pat. Nos. 3,853,465 and 4,184,848). Since only surfactants were used, a rather high concentration of surfactant was needed for an effective clearing. High concentration of surfactants are often interacted with other chemical or enzyme reactions and causes complication of analysis.
In the present invention, a relative low concentration of surfactant is used. This is accomplished by the action of enzyme (cholesterol esterase or lipase) added to the clearing reagent.
The exact mechanism of clearing by this invention is not yet known. It is possible to speculate however that, in cases where patients have hyperlipemia, the turbidity found in serum samples is due primarily to an elevated triglyceride content.
Triglycerides are water-insoluble, and usually buried inside the fat core with cholesterol esters in lipoprotein complex. The clearing of a lipemic sample must be brought about by the disrupture of lipoprotein by a surfactant such as lauric acid diethanolamide (DEA), followed by the hydrolysis of triglycerides by the enzyme base. The surfactant also aids the dissolution of fatty acids released therein. Without enzyme, there is no clearing.
It is the object of this invention to provide a reagent which is effective in clearing turbidity in biological samples and to a method of effecting same, particularly in cases where the sample is to be photometrically assayed or analyzed for a particular component such as cholesterol.